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, 16 (2), 985-992

Metformin Exerts Anti-Inflammatory Effects on Mouse Colon Smooth Muscle Cells in Vitro

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Metformin Exerts Anti-Inflammatory Effects on Mouse Colon Smooth Muscle Cells in Vitro

Ahmed Al-Dwairi et al. Exp Ther Med.

Abstract

Inflammatory bowel disease (IBD) is a chronic incurable condition characterized by relapsing inflammation of the gut. Intestinal smooth muscle cells (SMCs) are affected structurally and functionally during IBD due to excessive production of different inflammatory mediators. Metformin is a widely used antidiabetic agent known to exert several anti-inflammatory effects in different tissues independently from its hypoglycemic effect. The aim of the present study was to investigate the effect of metformin on expression and secretion of different cytokines and chemokines from mouse colon SMCs (CSMCs) following induction of inflammation with lipopolysaccharide (LPS) in vitro. CSMCs from male BALB/c mice were isolated and cultured in Dulbecco's modified Eagle's medium and treated with LPS (1 µg/ml) and 0, 5, 10 or 20 mM metformin for 24 h. Expression and secretion of tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α), macrophage colony stimulating factor (M-CSF), T cell activation gene-3 (TCA-3) and stromal cell-derived factor-1 (SDF-1) was evaluated by ELISA. LPS-treated CSMCs demonstrated significantly increased expression of TNF-α, IL-1α, M-CSF, TCA-3 and SDF-1 when compared with the control group (P<0.05). Co-treatment with metformin (5 and 10 mM) significantly reduced their expression by ~20-40% when compared with LPS treatment alone (P<0.05). Furthermore, secretion of TNF-α, IL-1α, M-CSF and TCA-3 into the conditioned media was significantly decreased by metformin (5 and 10 mM; P<0.05). In addition, metformin decreased levels of LPS-induced nuclear factor-κB phosphorylation. These data suggest that metformin may provide beneficial anti-inflammatory effects on CSMCs and it may be utilized as an adjunct therapy for patients suffering from IBD.

Keywords: inflammation; inflammatory bowel disease; metformin; smooth muscle.

Figures

Figure 1.
Figure 1.
Identification and viability of mouse CSMCs. (A) A single, spindle-shaped CSMC under phase contrast microscopy; magnification, ×20. (B) Cell viability assay for cells treated with LPS (1 µg/ml) plus varying amounts of metformin (0, 5, 10 or 20 mM). Scale bar=50 µm. LPS, lipopolysaccharide; CSMC, colonic smooth muscle cell; Met, metformin; OD, optical density.
Figure 2.
Figure 2.
Effect of metformin treatment on expression of inflammatory cytokines by mouse CSMCs, evaluated using ELISAs. Levels of (A) TNF-α, (B) IL-1α, (C) M-CSF, (D) TCA-3 and (E) SDF-1 in CSMCs treated with LPS (1 µg/ml) and varying amounts of metformin (0, 5, 10 and 20 mM). *P<0.01 vs. the Ctrl group; #P<0.01 vs. the LPS group; §P<0.05 vs. the LPS group. LPS, lipopolysaccharide; CSMC, colonic smooth muscle cell; Met, metformin; TNF-α, tumor necrosis factor-α; IL, interleukin; M-CSF, macrophage-colony stimulating factor; TCA-3, T cell activation gene-3; SDF-1, stromal cell-derived factor-1; Ctrl, control.
Figure 3.
Figure 3.
Effect of metformin treatment on secretion of inflammatory cytokines by mouse CSMCs into the conditioned media, evaluated using ELISA. Levels of (A) TNF-α, (B) IL-1α, (C) M-CSF and (D) TCA-3 in CSMCs following treatment with LPS (1 µg/ml) and varying amounts of metformin (0, 5, 10 and 20 mM). *P<0.01 vs. the Ctrl group; #P<0.01 vs. the LPS group; §P<0.05 vs. the LPS group. LPS, lipopolysaccharide; CSMC, colonic smooth muscle cell; Met, metformin; TNF-α, tumor necrosis factor-α; IL, interleukin; M-CSF, macrophage-colony stimulating factor; TCA-3, T cell activation gene-3; Ctrl, control.
Figure 4.
Figure 4.
Metformin reduces nuclear LPS-induced NF-κB phosphorylation. Total nuclear proteins were extracted and nuclear NF-κB p65 was assayed in the lysate in colonic smooth muscle cells following treatment with LPS (1 µg/ml) and varying amounts of metformin (0, 5, 10 and 20 mM) *P<0.01 vs. the Ctrl group; #P<0.01 vs. the LPS group. LPS, lipopolysaccharide; Met, metformin; NF-κB, nuclear factor-κB; OD, optical density; Ctrl, control.
Figure 5.
Figure 5.
Proposed model for the mechanism by which metformin may suppress LPS-induced inflammatory gene expression in mouse colonic smooth muscle cells. Metformin may activate AMPK, which may interfere with LPS-induced NF-κB activation, phosphorylation and translocation to the nucleus, which in turn suppresses inflammatory cytokine and chemokine expression. Curved arrows indicate downstream activation and triangles indicate metformin. LPS, lipopolysaccharide; NF-κB, nuclear factor-κB; AMPK, adenosine 5′-monophsphate kinase; TLR4, Toll-like receptor 4.

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